Cell signaling is crucial for survival; without it physically or chemically isolated cells undergo apoptosis. In cancer cells many contact dependent processes are aberrant, but it has been shown that reception of adrenomedullin-mediated signaling is an essential for survival of cells in 80% of tumors. While many hormones and cytokines bind to specific receptors, adrenomedullin (AM) acts through a receptor known as the Calcitonin Receptor Like Receptor (CRLR).
The calcitonin family of bioactive peptides comprises of calcitonin, amylin, two Calcitonin-Gene Related Peptides (CGRP1, and CGRP2) and adrenomedullin (AM). Calcitonin is 32 amino acid peptide found in the parafollicular “C” cells of the thyroid in mammals as well as in a number of non-mammals. Calcitonin regulates the mineral (calcium and phosphate) balance. Calcitonin causes hypercalcemia by acting as an inhibitor of osteoclast induced bone resorption. CGRP is a 37 amino acid peptide produced by tissue specific processing of the calcitonin gene. Calcitonin is the major product in the thyroid, whereas CGRP is the major product in neural tissues. CGRP is a potent cardiovascular agent and has structural similarity with amylin. CGRP is found in two isoforms (CGRP-I and CGRP-II) that differ only by 3 amino acids.
Adrenomedullin (AM) is a 52 amino acid hypotensive peptide. It has structural similarity with CGRP and amylin. AM is produced in peripheral tissues, adrenal medulla, lung, and kidney and it is unregulated in ischaemia. Receptors for AM exist in many tissues, for example in astrocytes in the central nervous system, in the iris muscle in the eye, in bone, blood vessels, the heart, kidney and skin (Uchikawa et al., Clin. Exp. Pharmacol. Physiol. 2005 August; 32(8):675-80; Sumanas et al., Blood. 2005 Jul. 15; 106(2):534-41; Cornish J, Reid J Musculoskelet. Neuronal Interact. 2001 September; 2(1):15-24; Yoshihara et al., Regul. Pept. 2005 Apr. 15; 127(1-3):239-44; Matsumoto et al., Clin. Exp. Nephrol. 2004 December; 8(4):316-21; Muller et al., Br J. Dermatol. 2003 January; 148(1):30-8). In general, the calcitonin family of peptides has N-terminal ring structures of 6-7 amino acids involving a disulfide and an amidated C-terminal end.
The calcitonin family of peptides act through G-protein coupled membrane receptors (GPCRs). The gene for calcitonin receptors has been cloned. It is homologous to GPCRs in family “B” which typically recognize regulatory peptides (secretin, glucagons, VIP). A homolog of the calcitonin receptor, the Calcitonin Receptor Like Receptor (CRLR, also known as CL) has been identified (human 461 aa; rat/mouse 463 aa) and has 55% homology with calcitonin receptor (Njuki et al., Clin. Sci. 85, 385-388 (1993); Chang et al., Neuron 11, 1187-1195 (1993); Fluhmann et al., Biochem. Biophys. Res. Commun. 206, 341-347 (1995); Kapas et al., J. Biol. Chem. 270, 25344-25347 (1995)). Two related members of the family “A” class of GPCR, RDC1 and G10D, were identified as receptors for CGRP and AM, respectively.
Alone, the CRLR is unable to transduce a signal in response to AM, as the presence of a RAMP (Receptor Activity Modifying Protein) is needed to induce ligand specificity, binding and activation of the CRLR. The RAMPs are family of small intrinsic membrane proteins, with a predicted sizes of 14,000-17,0000 Kd. RAMPs consists of approximately 120 amino acids with a large extra-cellular domains of around 100 amino acids; a single membrane spanning domain and a short intra-cellular region of approximately 10 amino acids.
It has been shown that CRLR can function as either a CGRP receptor or an AM receptor, depending upon which members of the RAMP family, RAMPs1-3, are expressed. RAMP1, 2 and 3 contain an N-terminal signal peptide, an extracellular N-terminus, a single transmembrane domain near the C-terminus, and cytoplasmic C-terminus. RAMP1-3 displays 31% identity. RAMP-2 and RAMP-3 have approximately 30% identity. RAMPS may be involved in the transport of CRLR to the plasma membrane.
The three members of the RAMP family, RAMP1, 2 and 3, engender different ligand specificities of the CRLR so that:
RAMP1+CRLR=CGRP receptor
RAMP2+CRLR=AM receptor
RAMP3+CRLR=AM receptor
RAMP1 presents CRLR at the plasma membrane as a terminally glycosylated, mature glycoprotein and a CGRP receptor, whereas RAMPs 2 and 3 present CRLR as an immature, core glycosylated ADM receptor (McLatchie et al., 1998).
The present invention relates to the identification of therapeutic agents with the ability to influence RAMP-CLRL interactions. Such agents are targets for, inter alia, cancer therapy.